Inheritance of 2,4-dichlorophenoxyacetic acid (2,4-D) resistance in Amaranthus palmeri

In this study, the inheritance of 2,4-D resistance in a multiple herbicide-resistant Palmer amaranth (KCTR) was investigated. Direct and reciprocal crosses were performed using 2,4-D-resistant KCTR and susceptible KSS plants to generate F1 progenies. 2,4-D dose–response assays were conducted to evaluate the response of progenies from each F1 family along with KCTR and KSS plants in controlled environmental growth chambers. Additionally, 2,4-D-resistant male and female plants from each of the F1 families were used in pairwise crosses to generate pseudo-F2 families. Segregation (resistance or susceptibility) of progenies from the F2 families in response to a discriminatory rate of 2,4-D (i.e., 560 g ae ha−1) was evaluated. Dose–response analysis of F1 progenies derived from direct and reciprocal crosses suggested that the 2,4-D resistance in KCTR is a nuclear trait. Chi-square analyses of F2 segregation data implied that 2,4-D resistance in KCTR is controlled by multiple gene(s). Overall, our data suggest that the 2,4-D resistance in KCTR Palmer amaranth is a nuclear inherited trait controlled by multiple genes. Such resistance can spread both via pollen or seed-mediated gene flow. In future, efforts will be directed towards identifying genes mediating 2,4-D resistance in KCTR population.

www.nature.com/scientificreports/ weed resistance to SAHs. As of 2021, globally, 42 weeds are resistant to SAHs, amongst, 25 weeds have evolved resistance to 2,4-D 1 . This limit options for managing dicot weeds in cereal cropping systems. Moreover, increased adoption of commercialized 2,4-D-tolerant crop technology poses the risk of increased 2,4-D selection pressure, in several weeds 9 . Inheritance of 2,4-D resistance has been studied in several economically important weeds around the globe, such as prickly lettuce (Lactuca serriola L.), wild radish (Raphanus raphanistrum L.), wild mustard (Sinapis arvensis), and oriental mustard (Sisymbrium orientale L.) 6,[10][11][12] . So far, the inheritance pattern of SAHs such as 2,4-D resistance has varied depending on the resistance mechanism and the weed species involved. For instance, 2,4-D resistance in prickly lettuce was governed by a single co-dominant allele 12 . Similarly, in wild mustard (Brassica kaber L.) a single gene was found to mediate 2,4-D resistance 10 . However, polygenic inheritance was documented for MCPA resistance in hemp-nettle (Galeopsis tetrahit L.) 13 .
Palmer amaranth (Amaranthus palmeri S. Watson), a native of Mexico and the Southern United States, is one of the most common and troublesome weeds in the United States 14 . So far, Palmer amaranth has evolved resistance to 8 herbicide sites of action (SOAs) 1 . The reproductive biology of Palmer amaranth plays a crucial role in its ability to evolve resistance to herbicides. For instance, Palmer amaranth is a dioecious plant, making cross-pollination a necessity to produce seeds 15,16 . Female Palmer amaranth plants are characterized by high fecundity resulting in the production of abundant seeds per plant 17,18 .
We documented a multiple resistant population of Palmer amaranth (Kansas Conservation Tillage resistant; KCTR) from Kansas which has evolved resistance to six herbicides, including 2,4-D 19,20 . Furthermore, recently we reported enhanced metabolism of 2,4-D in KCTR compared to KSS and MSS indicating metabolic resistance, even though research on possible co-existence of TSR is warranted 19 . Although resistance to 2,4-D in Palmer amaranth has been documented in two populations in Kansas [19][20][21] , the information on the inheritance of 2,4-D resistance is lacking. This study was conducted to investigate the inheritance of 2,4-D resistance in KCTR Palmer amaranth. The specific objectives were: i) generate F 1 progenies and evaluate their response to 2,4-D application, and ii) generate F 2 progenies and determine if 2,4-D resistance in KCTR is conferred by either single or multiple genes.

Results
2,4-D dose-response with KCTR, KSS, and F1 progenies. Response of progenies from F 1 families and parental Palmer amaranth populations, KCTR and KSS to 2,4-D was assessed via dose-responses experiments. Our data indicates that 90% of KCTR and 15% of KSS plants survived the discriminatory dose (1120 g ae ha −1 ) of 2,4-D. The KSS plants which survived 2,4-D treatment, showed severe epinasty (downward curling of plant parts, a typical symptom of auxinic herbicide injury) and stayed stunted and did not show any new growth up to 4WAT. Nonetheless, several KCTR Palmer amaranth plants survived even the highest dose used in the study (2240 g ae ha −1 ). Compared to that, 87% of F 1 -1, 92% of F 1 -2, and 89% of F 1 -3 survived application of 2,4-D at 1120 g ae ha −1 . The F 1 plants in all the three families, generated by both direct or reciprocal crosses survived 2,4-D application, implying that the resistance to 2,4-D in KCTR Palmer amaranth is a nuclear trait. In the F 1 -1 dose-response experiments, the amount of 2,4-D required to cause 50% reduction in growth (GR 50 ) of KCTR, F 1 -1 and KSS were 1800, 837, and 194 g ae ha −1 , respectively (Table 1; Fig. 1), suggesting that the KCTR and F 1 -1 are 9-and fourfold resistant to 2,4-D compared to KSS (Table 1). Similar results were obtained from F 1 -2 and F 1 -3 dose-response experiments (Tables 2, 3; Figs. 2, 3). The GR 50 of KCTR, KSS, and F 1 -2 plants were 1188, 501, and 200 g ae ha -1 , respectively (Table 2; Fig. 2), indicating that the KCTR and F 1 -2 plants were 6-and threefold resistant, respectively, to 2,4-D treatment compared to KSS. Likewise, the GR 50 of KCTR, KSS, and F 1 -3 were 1317, 500, and 167 g ae ha −1 , respectively (Table 3; Fig. 3), with 7 and 3 RI compared to KSS (Table 3). P-values from the t-test for each F 1 family are presented in supplementary material (Table S1). Overall, the results of 2,4-D dose-response experiments suggest an intermediate response of F 1 progenies compared to KCTR and KSS pla nts.
Further, cumulative dry weight data from the F 1 dose-response assays were plotted for KSS, KCTR, and F 1 plants at each 2,4-D dose (Fig. 2). The spread and distribution of dry weight of non-treated (NT; 0 g ae ha −1 ) KSS, KCTR, and the F 1 plants were similar (Fig. 2a). At doses up to 2× (1120 g ae ha −1 ), F 1 and KCTR plants had visually similar distribution (Fig. 2b-e), however, at 4× dose (4×; 2240 g ae ha −1 ), KCTR plants had a wider spread than F 1 plants (Fig. 2f), indicating higher level of resistance compared to F 1 progenies. Segregation (2,4-D resistance or susceptibility) of F 2 progenies. Response of progenies from F 2 families and KSS population (as control) were evaluated by treating with 2,4-D at the field recommended dose of 560 g ae ha −1 . Unfortunately, as mentioned in the methods, the KCTR plants were not used in this experiment along with KSS Palmer amaranth due to lack of enough seed and poor germination at the time of conducting this    www.nature.com/scientificreports/ experiment. As expected, upon treatment with 560 g ae ha −1 of 2,4-D, all KSS plants (n = ~ 50), either completely died or had dried leaves and stunted growth at 4 WAT (Table 4). Interestingly, the percent survival of F 2 progenies highly varied among families. The phenotypes of F 2 progenies ranging from high resistance (plants showing low epinasty and high dry weight accumulation to very low-level resistance (plants showing high epinasty and low dry weight accumulation) were observed (Fig. 4). In the first run of the segregation assay, all F 2 families deviated from the expected 3:1 (resistance:susceptibility) segregation ratio (Table 4). In the second run of the segregation assay, except for the F 2 -1 family, all other families did not follow the 3:1 (resistance and susceptible) segregation ratio (Table 4). Overall, when combined (run 1 and 2; Table 4) all F 2 -families failed to segregate as 3:1 (resistance and susceptible) ratio, indicating that more than one gene(s) is involved in mediating 2,4-D resistance in KCTR. www.nature.com/scientificreports/ The distribution of shoot dry weight per plant (g) of the F 2 progenies (n = 332) and KSS plants (n = 21) was illustrated through a density plot (Fig. 3). The distribution showed a wide spread of dry weight bins for F 2 progenies representing high plant to plant variation in response to 2,4-D treatment (Fig. 3).

Discussion
The evolution of 2,4-D resistance in Palmer amaranth has been reported in two populations, from Kansas, USA, including, KCTR [19][20][21] . KCTR has been documented to have evolved predominantly metabolic resistance to at least five herbicide SOAs including ALS-, photosystem (PS) II-, protoporphyrinogen oxidase-, 4-hydroxyphenylpyruvate dioxygenase-inhibitors and SAHs 20 . Information on the genetic basis of 2,4-D resistance in Palmer amaranth is lacking. Therefore, in the current study, we investigated the inheritance of 2,4-D resistance in KCTR.
In the dose-response assays, KCTR plants, as expected showed a high level of 2,4-D resistance (6-ninefold) compared to KSS plants (Fig. 1a-c; Tables 1, 2, 3). Progenies generated by both direct and reciprocal crosses were resistant to 2,4-D, indicating that the 2,4-D resistance in KCTR is a nuclear trait ( Fig. 1; Tables 1, 2, 3). In general,  www.nature.com/scientificreports/ resistance to majority of herbicides is a nuclear trait. One exception is the TSR to PS II-inhibitors in weeds, which is inherited maternally/cytoplasmically 22 . Nuclear inheritance of 2,4-D inheritance has been reported in several weeds, including wild radish, wild mustard, oriental mustard, and prickly lettuce 6,10-12 .  Tables 1, 2, 3) confirmed that 2,4-D resistance in KCTR Palmer amaranth is a nuclear trait and transferred to progenies generated from both direct and reciprocal crosses. Most cases of herbicide resistance, including TSR or NTSR, have been reported to be controlled by either completely or incompletely dominant genes 6,11,26,27 . The 2,4-D resistance in wild radish was found to be incompletely dominant 6 . In contrast, inheritance of 2,4-D resistance in wild radish and dicamba resistance in kochia (Bassia scoparia L.) were found to be mediated by highly dominant alleles 6,28,29 . Both dominant and incompletely dominant traits can be expressed in homo-or heterozygosity, therefore, the spread of 2,4-D resistance will be faster than a recessive trait.
Segregation of F 2 progenies in KCTR Palmer amaranth at the field recommended dose of 2,4-D (560 g ae ha −1 ) did not follow the Mendelian 3:1 (resistance: susceptible) ratio (expected for a single gene inheritance) ( Table 4). This indicates that the 2,4-D resistance in KCTR Palmer amaranth is a polygenic trait. Furthermore, the chi-square goodness of fit test data suggests variability in the phenotypes among the F 2 resistant progenies, which cannot be explained by a single major gene-mediated resistance (Fig. 3). Although majority of TSR is controlled by a single gene 30,31 , the inheritance of NTSR was reported to be conferred by both single and multiple genes 7,8,[25][26][27] . For instance, monogenic metabolic resistance to chlorsulfuron and atrazine was documented in common waterhemp (Amaranthus tuberculatus Moq. Sauer.), Palmer amaranth, and sorghum 7,20,26,27,32 . Polygenic inheritance was reported for mesotrione resistance in common waterhemp and Palmer amaranth 7,26,27,32 . Similar to our findings, MCPA resistance in hemp-nettle was found to be polygenic 13 . On the other hand, single-gene inherited 2,4-D resistance was reported in wild mustard, oriental mustard, prickly lettuce 10-12 . Traits inherited by multiple genes spread slower than those with single alleles.
Our previous work indicates that KCTR Palmer amaranth evolved metabolic resistance, possibly mediated by cytochrome P450 activity 19,20 . In other 2,4-D, resistant weed species, such as common waterhemp metabolic resistance mediated by P450 activity has been reported 33,34 . Increased expression of a P450 cluster (P450-81E8) was recently reported in 2,4-D-resistant common waterhemp populations from both Nebraska and Illinois 35 . We have reported increased expression of this gene in KCTR plants with metabolic resistance to tembotrione 36 . Therefore, there is a need to check expression levels of this gene in 2,4-D resistant KCTR plants. Our findings www.nature.com/scientificreports/ suggest more than one gene confers 2,4-D resistance in KCTR plants. Therefore, it is possible that a combination of P450s or other genes may be involved in metabolic resistance to 2,4-D in KCTR Palmer amaranth. Metabolic resistance to herbicides usually follows a three-step detoxification process, including several genes facilitating herbicide conversion, degradation, and transporting or compartmentalizing of degraded molecules into the vacuole of a plant cell. Metabolic resistance might also involve genes that help in plant recovery after herbicide treatment. More in-depth investigation is needed to decipher the genes involved in the metabolic resistance of 2,4-D in KCTR Palmer amaranth.

Materials and methods
Plant materials and growth conditions. 2,4-D-resistant (KCTR) and susceptible (KSS; Kansas Susceptible) Palmer amaranth populations were used in this study. KCTR population was originally collected in 2018 from a long-term conservation tilled field with continuous sorghum production in Riley County, Kansas 20 . Although the original field collected seed of KCTR was found resistant to multiple herbicides, including 2,4-D, we selected 2,4-D survivors by treating the plants with 2,4-D at 560 g ae ha −1 . These plants were kept in isolation under greenhouse conditions to produce next generation of seeds. This process was repeated for two generations so that the seed used in this study were more homogenous in nature with respect to 2,4-D resistance. KSS population is known to be susceptible to 2,4-D and was also collected from a nearby field in Riley County, Kansas and has been characterized previously 20,23,37,38 . Experiments to generate F 1 and F 2 progenies for this study were conducted either in greenhouse or controlled environmental growth chambers. The following greenhouse conditions were maintained: Palmer amaranth seedlings with 3-4 leaves were transplanted into small pots (6 × 6 × 6.5 cm) for herbicide treatment. Plants were watered as needed (procedure for herbicide treatment is given later). Before enclosing the plants, existing flowering parts were removed to ensure crossing between selected plants.

Generation of F 1 and F 2 families of KCTR
Once both plants flowered, male Palmer amaranth plants were gently shaken to disperse pollen and pollinate the stigmas of female plants. At maturity, seeds were collected from each female plant separately, cleaned, and stored at 32 °C in greenhouses to allow over-ripening of the seeds necessary for good germination. In total three F 1 families were generated from these crosses, i.e., F 1 -1, F 1 -2, and F 1 -3 (Fig. 5). Plants from each F 1 family were further screened with 2,4-D (1120 g ae ha −1 ) to identify female and male F 1 plants resistant to 2,4-D to be used to generate pseudo-F 2 families (Fig. 5). In total four F 2 families were generated from these crosses (F 2 -1, F 2 -2, F 2 -3-1, F 2 -3-2; Fig. 5). These progenies from the F 1 and F 2 families were used in further studies.

2,4-D dose-response study using KCTR, KSS, and F 1 progenies. Whole-plant dose-response
experiments were conducted with parental KCTR, KSS Palmer amaranth plants, and F 1 progenies generated from three F 1 families. The experiments were conducted in a completely randomized design in growth chambers. KCTR, KSS, and progenies from individual F 1 families were germinated in small trays and transplanted into small pots as described previously. One week after transplanting, the seedlings were transferred to growth chambers (maintained at the same conditions as described in ('Plant Materials and Growth Conditions'). Palmer amaranth seedlings (10-12 cm tall) were treated with 2,4-D at the following doses i.e., 0 (non-treated), 140, 280, 560 (1×; field recommended dose), 1120, and 2240 (4×) g ae ha −1 . Thirty minutes after herbicide treatment, plants were returned to the growth chamber. 2,4-D was applied using a bench-track sprayer (Generation III, DeVries Manufacturing, Hollandale, Minnesota, USA) using a flat-fan nozzle tip (8002 Teejet, Spraying Systems Co., Wheaton, Illinois, USA) and was calibrated to spray equivalent to 187 L ha −1 (20 GPA). At 4 WAT, plant survival (dead or alive) was assessed at each dose of 2,4-D treatment. Palmer amaranth plants that survived 2,4-D dose (560 g ae ha −1 and above) and continued to grow were considered as resistant and those that showed severe epinasty (downward curling of plant parts, a typical symptom of synthetic auxin herbicides) and ceased to grow, were grouped as susceptible to 2,4-D. In response to 2,4-D treatment the plants that survived and Above-ground plant parts were harvested at 4 WAT and oven-dried at 65 °C for at least 72 h. Dose-response experiments with each F 1 family were repeated once, and 3-5 replications were maintained for each dose of 2,4-D. Dry weight was measured and converted into relative dry weight (% of non-treated) as described below (Eq. 1): (1) Relative dry weight (% of non-treated control) = Dry weight of the sample × 100 Average dry weight of non-treated control www.nature.com/scientificreports/ Evaluation of segregation (2,4-D resistance or susceptibility) of F 2 progenies. Seed of F 2 -families (F 2 -1, F 2 -2, F 2 -3-1, F 2 -3-2) were planted and the seedlings were grown in the same greenhouse as described above. Based on the response of parental populations i.e., KCTR and KSS in greenhouse conditions, the field recommended dose of 2,4-D (560 g ae ha −1 ) was identified to discriminate resistant (alive) or susceptible (dead) plants. The same dose was used to determine the segregation of F 2 progenies into resistant or susceptible plants. About 20-120 progenies (10-12 cm tall; Table 4) representing each F 2 family were treated with 560 g ae ha −1 of 2,4-D. About 20-30 plants of KSS Palmer amaranth were also treated along with the F 2 progenies as a negative control. Unfortunately, the KCTR plants were not included in this assay due to lack of enough KCTR seed and poor germination at the time of conducting this experiment. At 4 WAT, live or dead (resistance or susceptible) phenotype of the 2,4-D-treated plants was documented (Fig. 5). The experiment was repeated. For the second run of the experiment, at 4 WAT, the above-ground dry weight of F 2 plants was harvested, dried, and measured (as described above).

Data analysis.
Relative dry weight data from two runs of 2,4-D dose-response experiments of each F 1 family was compared using Levene's test (α = 0.05) in R (version 4.0.3, R Core Team, 2020) in an R-studio interface.
Since it was non-significant, dry weight data were pooled across two runs. Dry weight datasets for each F 1 family were analyzed using non-linear log-logistic three-parameter 'drc' models in R 39,40 . The three-parameter regression model used the following formula (Eq. 2): where Y is the response variable, x is the herbicide dose, d is the upper limit, b is the relative slope around e and e is GR 50 (amount of 2,4-D required to reduce plant dry weight by 50%). The GR 50 values of different F 1 families and populations were further tested via t-test using the function 'compParm' in 'drc' package to determine differences across the doses. In-built 'plot' function in the 'drc' package was used to visualize the dose-response curves. Resistance index (RI) was calculated as a ratio of GR 50 of KCTR or F 1 families and GR 50 of KSS population. Cumulative dry weight distribution of the F 1 progenies along with the parental populations (KSS and KCTR) at each dose of 2,4-D was illustrated (as density plot) using 'ggplot2' package in R 41 . For the plot, dry biomass was represented as bins (each bin represents 0.25 g plant biomass) and proportion of total plants having biomass in that bin was plotted as density.
(2) Y = d + exp b log x − log e www.nature.com/scientificreports/ The number of F 2 progenies observed to have survived after 2,4-D treatment was compared with the expected number of survivors using chi-square analyses. The expected segregation of 2,4-D resistant (alive) or susceptible (dead) phenotypes was determined based on the Mendelian single locus segregation model, which suggests that the 2,4-D resistance in KCTR is controlled by a single gene. The following Eq. (3) was used to test this assumption.
In this equation Exp. F 2 is the expected segregation frequency of the F 2 progenies and Obs. is the observed segregation frequency of KCTR, F 1 , and KSS phenotypes. Based on previous experiments conducted in greenhouse conditions, the KCTR plants and F 1 progenies were found resistant to 560 g ae ha −1 of 2,4-D, while KSS plants were susceptible. This dose was used to determine resistant or susceptible plants among F 2 progenies. Chi-square goodness of fit test (χ 2 ) was used to analyze the data of the plant segregation as resistance or susceptible to 2,4-D by comparing the observed segregation frequency with the expected. The null-hypothesis (H 0 ) for the test was that segregation of 2,4-D resistance in F 2 progenies will follow the 3:1 ratio (resistant: susceptible) and p-value from each F 2 family was compared with the significance level of α = 0.05. The formula used for the test is (Eq. 4): where χ 2 is the Chi-square goodness of fit-value, O and E are the observed and expected segregation frequencies, respectively, of progenies in each F 2 family. H 0 was rejected if the obtained p-value was < 0.05. Dry weight data of the F 2 progenies were also visualized as density plot (each bin represents 0.1 g plant biomass).
Permissions required. All the seed used was collected by the authors from the experimental field maintained by Dr. Peterson, one of the authors of this manuscript.
Guidelines required. The collection of Palmer amaranth seed used in this study complies with the institutional guidelines.

Conclusion
Results of this study suggest that 2,4-D resistance in KCTR Palmer amaranth is a nuclear trait, governed by multiple genes. The possible presence of TSR to 2,4-D in KCTR has not been investigated yet and will be interesting future line of work. Future research will also be aimed at discovering metabolic genes that mediate 2,4-D resistance in KCTR. Availability of Palmer amaranth genome 42 and application of transcriptomic approach can enable identifying specific P450s involved in metabolic resistance in weeds. For example, a P450 gene (P450-81A10v7) was documented to confer metabolic resistance to at least 5 SOAs in rigid ryegrass (Lolium rigidum L.) 43 . Studies directed to investigate if metabolic resistance to 2,4-D is transferred via pollens to other Amaranthus sp. can help in formulating management practices specific to Amaranthus sp. Overall, 2,4-D has been a viable option to manage ALS-and EPSPS-inhibitor resistant Palmer amaranth; moreover, 2,4-D resistant crop technology has been shown to provide growers with options to effectively manage glyphosate-resistant Palmer amaranth 44 . However, the evolution of resistance to 2,4-D limits the use of this important herbicide by growers. Therefore, the incorporation of integrated weed management techniques will be necessary for reducing the evolution and spread of 2,4-D resistance.

Data availability
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.